This invention relates to a method for decreasing intestinal cholesterol absorption in man and, specifically to inhibiting or decreasing intestinal cholesterol and fatty acid absorption by oral administration of non-absorbable synthetic sulfated polysaccharides which inhibit the pancreatic cholesterol esterase catalyzed hydrolysis of naturally occurring and ingested cholesterol esters. The invention is based upon our discovery that cholesterol esterase is a more important contributor to overall dietary cholesterol absorption than previously accepted because of our surprising finding that (1) cholesterol derived from cholesterol esters is preferentially absorbed compared to free cholesterol and (2) cholesterol esterase enhances the absorption of free cholesterol. Thus, the surprising usefulness of inhibiting cholesterol esterase has demonstrated a new need for potent (Ki less than 5 .mu.M) inhibitors of cholesterol esterase. The invention is based on our discovery and synthesis of non-absorbable, non-degradable sulfated polysaccharides which are potent inhibitors of human pancreatic cholesterol esterase, the enzyme responsible for promoting the intestinal absorption of cholesterol and fatty acids. This invention is also based on our observation that such agents are stable and bio-available to the intestine when delivered in baked goods such as biscuits and can therefore be administered in food products. This invention is also based upon our discovery that another class of potent non-absorbable inhibitors of cholesterol esterase are antibodies to cholesterol esterase.
Atherosclerosis is the leading cause of death in the United States and high serum cholesterol concentrations are associated with increased risks of fatal artherosclerotic events, JAMA, 1985, 253: 2094 (NIH Consensus Panel Report). In 1988, a Consensus Panel of experts at the NIH stated that a major public health priority was the reduction of cholesterol, and that the goal of front line therapy should be to diminish the intestinal absorption of cholesterol, either through eating less cholesterol or through the use of drugs which act in the intestine to reduce cholesterol levels, Arch. Int. Med., 1988, 148: 36 (Consensus Full Report). However, no mention was made of the importance of dietary cholesterol esters or attempts to lower cholesterol by inhibiting cholesterol esterase and no drugs now exist that block cholesterol absorption from the intestine. Currently, the principal drug to act within the intestine to lower cholesterol is cholestryamine, a bile acid sequestrant, "Agents to Treat Hyperlipidemia", The AMA Drug Evaluations, 6th Ed., p. 903. This agent binds bile salts within the intestinal lumen, and the resulting complex is excreted in the feces. Since bile acid is not re-absorbed, the liver uses additional cholesterol to synthesize more bile acid which effectively lowers the sterol concentration in the body. Bile salt sequestrants are etfective in lowering cholesterol, but they seldom reduce cholesterol by more than 15%, and they are poorly tolerated by patients. Large quantities of these ion exchange resins must be ingested (15g or more), which lead to assaults on both the gustatory senses and intestinal function. Common side effects are constipation and bloating, JAMA, 1985, 253: 2095.
Cholesterol esterase is secreted by the pancreas after eating and is active in hydrolyzing ingested dietary esters of cholesterol. No role for it is established in the absorption of free cholesterol, but the enzyme has been regarded as essential for absorption of cholesterol derived from cholesterol esters. If enzyme activity is removed from pancreatic Juice, no cholesterol absorption from cholesterol oleate occurs. If the cholesterol esterase activity is returned, absorption of cholesterol occurs, Borja et al., 1964, J. Physiol 206:223 and Vahouny and Treadwell, 1964, Proc. J. Exp. Biol. and Med. 116: 496. Since fatty acids that are absorbed come in part from cholesterol esters and they contribute to atherosclerosis, the enzyme cholesterol esterase may enhance atherosclerosis in two ways.
Despite this information and the stated mission of the NIH to target strategies of diminishing cholesterol absorption from the intestine, no study of pharmacological inhibition of human pancreatic cholesterol esterase has been performed. In fact, few studies have focused on the human enzyme at all, with most attention directed to other mammalian enzymes (rat, pig and cow) Calame et al., 1975, Arch. Biochem. Biophys. 168: 57; Van den Bosch et al., 1973, Biochem. Biophys. Acta 286: 94; Momsen et al., 1977, Biochem. Biophys. Acta 486: 103; Guy et al., 1981, Eur. J. Biochem. 117: 457; Slatton et al., 1986, Biochem. Biophys. Res. Comm. 134: 386; and Borgstrom, 1988, Biochem. Biophys. Acta 962: 308. Only one study has shown that a cholesterol esterase inhibitor can decrease cholesterol absorption in animals, Fernandez et al., 1989, Biochem. Biophys, Acta 1001: 249. However, this agent is a weak inhibitor of another activity of cholesterol esterase (Ki 100 .mu.M), involving water soluble substrates and other reports claim that it does not inhibit cholesterol esterase at all, Hadvary et al., 1987, Int. J. Obesity 11: Suppl. 2, 21. In addition, this agent is 30% absorbed and metabolized. Thus, the lack of an appreciation of the importance of cholesterol esters in contributing to the overall absorption of cholesterol from the diet has prevented the development of inhibitors of cholesterol esterase, and no focus on non-absorbable inhibitors has been achieved.
Because of our discovery of the preferential absorption of these esters and the unexpected observation that cholesterol esterase can stimulate absorption of free cholesterol, there is now an important need to develop inhibitors of human pancreatic cholesterol esterase, especially those that are potent (Ki less than 5 .mu.M), non-absorbable and non-degradable. The pharmacology of various sulfated polysaccharides has been investigated. Cook and Cammarata, 1963, Arch. Int. Pharmacodyn. 144: 1. In particular sulfated amylopectin has been taught in U.S. Pat. No. 4,150,110 as an anti-ulcer agent, but its properties as a cholesterol esterase inhibitor, which decrease absorption of cholesterol, have not been recognized. Sulfated dextran of low molecular weight has been recognized for use in the treatment of hyperlipemia and as an orally administered anticoagulant, British Patent No. 953,626. These sulfated dextrans, of low molecular weight (7000-8000, 7-8 kDa) developed for their enhanced absorption characteristics from the intestine, have been shown to reduce serum cholesterol levels at a dose of 1800 mg/day, Goro et al., 1987, J. Clin. Biochem. Nutr. 2: 55 by activating a blood enzyme, liproprotein lipase. However, their weak ability to inhibit cholesterol esterase or co-inhibit cholesterol absorption has not been recognized. They also have not been used as food additives. High molecular weight dextran sulfate has been excluded from development by others because of its lack of absorption and its attendant lack of activating serum lipoprotein lipase. Low molecular weight dextran sulfates because of their absorption cause changes in the integrity of the blood clotting system in man, a dangerous side effect in chronic usage, Drug In Japan (Ethical Drugs 10th ed. (1986) and see also product insert for MDS Kowa Tablet, Kowa Co., Nagoya Japan.